hello friends ;-)
I'm in the process of designing an oligonucleotide pair to get an adapter
with 4bp-overhangs on each end... I have a designed a couple of oligos with the
desired ends and got kcal/mol values for each 13-mer from primer analysis
software... There is a stable dimer of 6 bp (plus one base further another
single base match) with -10.3 kcal/mol and because one overhang is for NotI RE
the GGCC 3'-dimer has also a high value of -9.3 kcal/mol...
In order to get an idea about the value for the desired 9 bp dimer I used the
program FoldRNA in GCG with the seq written from 5' -> 3' and a stretch of
"N" inserted in between... I had seen something like this in a Biotechniques
article a few years ago where 10 NNNNNNNNNN had been used to make a circular
[RNA] molecule to check out for primer dimers...
The obtained value was -12.3 kcal/mol... however, the value increases when I
reduce the number of Ns and is for instance -13.6 kcal/mol if 6 are inserted
instead of 10... if 50 Ns are inserted it can be about -9 kcal/mol... and if
no Ns are inserted [ hence 4 base overhang loops directly back onto stem!] I
read -12.0 kcal/mol...
Well, now I don't know which [if any] of those approximations I can accept...
and I would be indeed very grateful, if somebody could let me know how I could
calculate a suitable value... I suspect that during slow cooling of the heated oli
mixture the designed molecule should perform properly even if a slight risk
remains for those +6 bp stretches versus the "full-length" 9 bp duplex with
4-base overhangs...
Thank you very much for any insights - I highly appreciate!
Have a nice time,
Roland